Methods and apparatus for handling microbial samples

ABSTRACT

This invention pertains to the general field of microbiology, and more specifically to transfer, inoculation and/or streaking of micro-organisms, e.g. for the purpose of obtaining individual colonies. Provided is a method for transferring a microbial sample from a first carrier to a second carrier, comprising the steps of:
         a) contacting at least one ferromagnetic particle with at least part of the sample associated with the first carrier, wherein the particle is a composite bead having a surface roughness (Ra) in the range of 0.1 to 25 μm, a diameter between 2 to 88 mm and a density below 7 g/cm 3 , and   b) applying a magnetic field gradient to allow for magnetically controlled motion of said particle to said second carrier, such that at least part of the sample is streaked onto the second carrier. Also provided is an apparatus for inoculating petri dishes with the sample according to the method and also inoculating slides and/or tubes with a portion of the sample. A container that receives the magnetic composite bead that carries samples from the container when removed is also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/740,746, filed on Sep. 13, 2010, which application is a nationalphase entry under 35 U.S.C. § 371 of International Application No.PCT/NL2008/050693 filed Nov. 4, 2008, which claims priority fromEuropean Patent Application No. 07119955.8 filed Nov. 5, 2007, all ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention pertains to the general field of microbiology, and morespecifically to transfer, inoculation and/or streaking ofmicroorganisms, e.g. for the purpose of obtaining individual colonies.Among others, it relates to methods for handling and streaking microbialspecimens and to a (semi-) automated apparatus to prepare specimens forvisual counting, typing and other analysis of bacterial populations, forexample in diagnostic microbiology.

One of the many procedures, which must be performed in microbiology, isplate streaking with the purpose of isolating microbiological colonies.The isolated colonies are absolutely necessary for the observance ofcolony morphology and for the performance of staining and otherprocedures that are necessary for determining the genus and in manycases, the species, strain, etc. of an unknown organism. In the classictechnique, known as streaking, a sterile loop is first brought intocontact with the sample or inoculum whose cells one wishes to transfer;it is then brought into contact with the plate containing sterile solidmedium to which one wishes to transfer the cells, and is moved back andforth over the surface of a portion (e.g. one quarter) of the freshplate. In the initial step, millions of cells may be transferred to thenew plate, and must be further diluted by many orders of magnitude sothat individual colonies, rather than confluent growth, will beobtained. Since the loop surface is also now contaminated with manythousands of cells, it must be either resterilized (e.g., by flamesterilization in the case of wire loops), or substituted by a newsterile loop (e.g. in the case of single-use, disposable plastic loops).The sterile loop is then brought into contact with the surface of thefresh plate which has been previously streaked, e.g. by severalmovements across the zone of the first streaking, and then anotherportion of the plate (e.g. a quarter of the plate adjacent to the firststreaking) is streaked. This results in additional dilution of thecells. The loop is then either resterilized or replaced, is brought intocontact with the second streaking area, and then a third zone of theplate is streaked. This process must be performed at least three or fourtimes in order to be certain of obtaining individual colonies, ratherthan confluent growth, following incubation of the fresh plate.

The testing of clinical microbial specimens typically requires thestreaking of several (e.g. 2 to 6) plates per test specimen. The manualstreaking process requires approximately 30 seconds per agar plate of askilled technologist's time. The quality of the streak and therefore thedegrees of isolation of the micro-organism depends upon the trainingreceived by the technologist and the care taken in performing theprocess.

Thus, the manual inoculation and streaking process is verylabour-intensive and time-consuming. Furthermore, in many diagnosticsettings, such as a hospital microbial laboratory, there is often a peakload of samples coming in to be tested. It is not uncommon that theprocessing of up to a 1000 individual clinical samples of various typesmust be accomplished in a time window of only a few hours, typically atthe end of a normal working day, like between 3 and 5 μm. This puts anenormous time pressure on the employees, equipment and laboratory spaceinvolved. The inoculation and streaking is however only the beginning ofthe actual microbial testing, and peak loads are also encountered fordownstream procedures, including plate incubation, processing (e.g.staining) and the final plate assessment.

Therefore, there have been attempts in the field to automate the plateinoculation and/or streaking process.

For example, the ISOPLATER 180 (Vista Technology Inc., Edmonton,Alberta, Canada) is an automated Petri dish streaker. The machineautomatically rotates the load carousel to bring in a stack of plates,downloads a dish, removes the lid, orients the dish, transfers the dishwith its lid, streaks in four successive quadrants for isolation,replaces the lid and uploads the completed dish into the unloadcarousel. Spreading over the entire surface of an agar plate isaccomplished by four individual nichrome loops which are sterilized byelectrical heating between dishes.

WO2005/071055 discloses a streaker device and apparatus for streaking amicrobial inoculum for single colonies on the surface of a solid growthmedium. The device is characterized by a “comb-like” structureconsisting of a row of spaced apart contact surfaces that areresiliently supported by a common support member. The streaking deviceis applied to the surface of an agar plate and is rotated to variousdegrees. Prior art dating back to the early eighties discloses streakingsystems comprising the use of a spherical, magnetic metal particle, inparticular steel balls. For instance, U.S. Pat. No. 3,830,701 describesa method and an apparatus for streaking a microbial sample comprisingthe contact of a stainless steel ball with an inoculum and generating acontrolled motion of the ball using a moving magnet to transfer themicrobial sample to a culture surface. U.S. Pat. Nos. 3,660,243 and3,623,958 disclose methods and devices comprising similar features.

The systems known in the art will automate and replace many of themanual tasks traditionally involved in the inoculation and streaking ofstandard agar plates. However, they do not accommodate all of the user'spreferences. These include: a) production of single colonies when usingminiaturized or split plates; b) no cross-contamination; c) ease of use;d) large capacity (preferably at least 500 plates/hour); e) low cost andf) compatibility with different specimen types, e.g. liquid samples,swabs etc. and non-standard plates, such as split-plates.

BRIEF SUMMARY OF THE INVENTION

The present inventors set out to address at least some of the abovepreferences in an attempt to provide further improved methods andapparatus for (semi-) automated handling of a microbial sample. Theyobserved that the steel particles employed in the magneticallycontrolled streaking methods known in the art are unsuitable forreliable, high-speed automated streaking that is sought for today. Inparticular, it was found that the steel balls known in the art displayinsufficient rolling to achieve a good spreading of the inoculum onto a(culture) surface. At the high streaking rates desired nowadays (atleast 500 plates/hour), the steel particles were ineffective to yieldsingle colonies, especially when more complex streaking patterns wererequired to obtain a sufficient streaking path length on a limitedsurface area, e.g. the zigzag pattern with sharp bends to streak sampleson split plates. Without wishing to be bound by theory, it is believedthat a homogenous, non-composite steel particle forms a dipole and thusacts as a magnet itself. As a result, the steel particle “slips” on thesurface of the plate during magnetically controlled motion and theparticle cannot make optimal rolling movements.

It was surprisingly discovered that much better streaking results couldbe obtained when a non-homogenous, composite magnetic particle is usedinstead of the homogenous stainless steel particles known in the art.Furthermore, other particle characteristics such as diameter, densityand/or surface roughness were identified as relevant for spreadingperformance of the particle. As is exemplified herein below, it wasfound that contacting a sample with a magnetic composite particle, suchas a magnetic bead comprising both a magnetic and a non-magneticmaterial, allows for a very efficient and easy transfer of various typesof samples from one carrier to another, e.g. from a urine collectiontube or swab to a broth, a Petri dish or a microscopic glass slide.Furthermore, it was surprisingly observed that a microbial sample can bespread onto a solid surface or homogenized in a liquid medium by themotion of the magnetically controlled composite particle. The spreadingonto a solid growth medium by placing a magnetic field under it producedmore single, isolated microbial colonies when compared to manualstreaking, even at high speed. A predetermined streaking pattern can beobtained with optimal usage of the surface area of a plate. The totalpath length of a streak can thus be increased within the confines of theplate. This obviates the need to switch to a larger size, and moreexpensive, culture plate. Of course, the longer the streak path length,the better the chance that an inoculum is sufficiently diluted to obtainisolated colonies. An optimal utilization of the plate surface area isespecially advantageous when using miniaturized plates or so-calledsplit plates having two different media contained in separatecompartments of a single plate.

A further advantage of the magnetically controlled streaking as providedherein is that it can maximally accommodate surface variation. Incontrast to the machine-held streaking devices known in the art, themagnetic particle used for sample transfer or streaking is notmechanically restrained in any fashion. It can therefore smoothly followany surface without the risk of damaging the surface of e.g. a solidgrowth medium.

The invention therefore provides a method for transfer of a microbialsample, preferably a liquid sample, from a first carrier to a secondcarrier, said “transfer method” comprising the steps of:

a) contacting at least part of said sample associated with said firstcarrier with at least one ferromagnetic particle, the particle being aspherical composite bead; and

b) applying an external magnetic field gradient to allow formagnetically controlled motion of said bead, such that at least part ofthe sample is transferred to said second carrier.

Also provided is a method for streaking a microbial sample onto a solidcarrier, said “streaking method” comprising the steps of:

a) contacting at least one ferromagnetic particle with a surface of thesolid carrier, the particle preferably being a spherical composite bead,followed or preceded by providing the particle with at least part ofsaid sample, and

b) applying an external magnetic field gradient to allow formagnetically controlled motion of said particle on said surface, suchthat at least part of the sample is streaked onto the solid carrier.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1, 1A and 1B show schematic perspective views of a solid carrierprovided with a magnetic particle. Sample is not included in thedrawings. Illustrates are various embodiments wherein a two-dimensionalmagnetic field gradient may be used to control the motion of a magneticparticle, in this case a magnetic bead on the surface of a solidcarrier, e.g. a growth medium contained in a Petri dish. In FIGS. 1A and1B, a section of the Petri dish has been cut away to better illustratethe position of the external magnet(s).

In FIG. 1, the movement of a magnet particle 1 on the surface of dish 3is controlled through a magnetic field gradient generated by permanentmagnet 2 attached to a movement mechanism 4, capable of moving themagnet in the x- and/or y-direction. The movement mechanism may becontrolled by software to generate a magnetic field gradient to createthe desired streaking pattern.

In FIG. 1A, the magnetic bead 1 is moved across the agar surface by useof a controllable electromagnet 102. The electromagnet 102 is positionedjust below the Petri dish 3. The magnet can be moved using the mechanismshown in panel A.

In FIG. 1B, the movement of the magnetic bead 1 is controlled through agrid of multiple adjacent electromagnets 202. The electromagnet gridmoves the bead through switching the magnets on and off in controlledpattern and order, thereby generating a magnetic field gradient.Herewith, the bead can be moved on the surface of dish 3 without theneed of a movement mechanism having movable parts.

FIG. 2 shows schematic drawings of exemplary streaking patterns that canbe obtained using a method of the invention.

Panel A: zig-zag streak pattern on whole plates extensive

Panel B: zig-zag streak pattern on whole plates intensive during firsthalf of the plate and then extensive on second half of the plate

Panel C: zig-zag streak pattern on split-plate

Panel D: four quadrant streak pattern on whole plate

Panel E: at random movement

Panel F: streak on glass slide

FIG. 3 shows schematic drawings of the cross-section of the variousbeads used in Example 2. Bead number 1 is a non-composite steel beadknown in the art. Beads 2-5 represent composite beads according to theinvention. For details see Example 2.

MICROBIAL SAMPLE

As used herein, the term “microbial sample” refers to any sample orspecimen known to contain, or suspected to contain, a microorganism or amixture of microorganisms. Preferably, the microbial sample is a liquidsample.

In one embodiment, it is a culture of microorganisms for reference andresearch. In order to maintain strains of individual microorganisms, thecultures are usually streaked onto plates containing solidified medium.The inoculum of the culture is generally from a single colony on apreviously inoculated plate, and the aim of the streaking procedure isto obtain individual colonies of the strain on the fresh plate.Individual colonies of identical form signify to the worker that asingle cell type has been transferred; moreover a single colony servesas the inoculum for further strain transfer and experimentation.

In another embodiment, a microbial sample is a sample for diagnosticmicrobiological analysis. Diagnostic microbiology is performed inmedical, environmental and food testing laboratories and hastraditionally used minimal process automation. Conventional laboratorydiagnostic microbiology involves specimen collection, registration,inoculation, isolation, identification and testing of pathogenicmicrobes from clinical specimens. The aim is to obtain isolated coloniesof microbes for subsequent analysis.

In a specific embodiment, a microbial sample is a clinical microbialspecimen. The isolation and identification of micro-organisms formedical or veterinary diagnostic purposes plays an important role indetermining which treatments are required by human or animal subjectswith various diseases. Exemplary clinical specimens that areadvantageously employed in a method of the invention include urine,upper respiratory swab, genital secretion, sputum, stool, pus, sterilebody fluid, and blood culture.

Magnetic Particle

The present invention provides the use of at least one ferromagnetic(also referred to herein as “magnetic” unless indicated otherwise)particle for the transfer, inoculation and/or streaking of a microbialsample. The term “ferromagnetic” as used herein is a broad term and isused in its ordinary sense and includes, without limitation, anymaterial that easily magnetizes, such as a material having atoms thatorient their electron spins to conform to an external magnetic field. Itis also referred to as “magnetically responsive material.” Ferromagneticmaterials include materials, such as metals, that are attracted topermanent magnets. Ferromagnetic materials also include electromagneticmaterials that are capable of being activated by an electromagnetictransmitter. In one embodiment, the at least one magnetic particlecomprises iron, nickel, cobalt or alloys thereof.

The particle may be solid, or semi-solid, e.g. having a hollow core. Forexample, it comprises a core material covered with a coating material.Factors that may be taken into consideration when choosing a particlematerial include inertness to microbial sample, resistance to (heat)sterilization, costs, etc. To facilitate efficient coating of theparticle with aqueous biological sample, it is preferred that thesurface is hydrophilic or at least non-hydrophobic. The particle can bere-used or it can be disposed after use.

Preferably, the particle is a bead. For streaking purposes, a compositebead is used. A bead is an essentially spherical particle. The term“composite” is meant to indicate that the bead is made up of two or morematerials, at least one of which is magnetic. Preferably, a compositebead of the invention comprises a magnetic and a non-magnetic material.In one embodiment, the composite bead comprises at least onenon-magnetic material, preferably a polymer, and at least one magneticmaterial. For example, the bead comprises a magnetic core (solid, wire)provided with a polymer coating. The core may have any suitable threedimensional structure. It is for example a solid, spherical core made ofa magnetic material. Alternatively, the magnetic core can have a morerandom structure, such as a tangled wire. In a specific embodiment, thecomposite bead is a steel bead covered with a hydrophilic material, suchas a hydrophilic polymer.

In another embodiment, the bead comprises a polymer core covered with amagnetic coating. Again, any core shape is encompassed. The magneticcoating may comprise particles of one or more magnetic materials, likeiron powder.

In yet another embodiment, the bead comprises or consists of ahomogeneous mixture of at least non-magnetic material, preferably ahydrophilic polymer, and at least one magnetic material in particulateform, preferably a powder. Very good results were obtained when therelative weight ratio of magnetic material to non-magnetic material isfrom between 10:90 and 30:70, preferably between 20:80 and 25:75. Anysuitable particulate magnetic material may be used. It is for examplemagnetite, ferrite or a mixture thereof.

Magnetite is a ferrimagnetic mineral with chemical formula Fe₃O₄, one ofseveral iron oxides and a member of the spinel group. The chemical IUPACname is iron(II,III) oxide and the common chemical name ferrous-ferricoxide. The formula for magnetite may also be written as FeO.Fe₂O₃, whichis one part wüstite (FeO) and one part hematite (Fe₂O₃). This refers tothe different oxidation states of the iron in one structure, not a solidsolution.

Ferrites are a class of chemical compounds with the formula AB₂O₄, whereA and B represent various metal cations, usually including iron. Theseceramic materials are used in applications ranging from magneticcomponents in microelectronics. Ferrites are a class of spinels,materials that adopt a crystal motif consisting of cubic close-packed(FCC) oxides with A cations occupying one eighth of the octahedral holesand B cations occupying half of the octahedral holes. The magneticmaterial known as “ZnFe” has the formula ZnFe₂O₄, with Fe3+ occupyingthe octahedral sites and half of the tetrahedral sites. The remainingtetrahedral sites in this spinel are occupied by Zn²⁺.

To allow for heat sterilization of a magnetic particle, it is preferredthat the materials used are mechanically resistant up to about theautoclave temperature of 120° C. Other beneficial properties includehigh chemical resistance, hydrophilicity, mixable with magneticparticulate material, chemical inertness, low costs, and ease toproduce. Suitable polymers for use in a composite bead of the inventioninclude epoxy resin, polyamide, polypropylene, and the like.

For mere sample transfer, any three dimensional shape capable oftransferring at least part of the sample can be used. It is for examplea spherical bead, a rod, an egg-shaped particle, a cubical particle or aparticle of undefined three dimensional shape. For an optimal streakingprocess it is important that the particle can “roll” (rather than slideor slip) on the solid surface along at least one axis of symmetry.accordingly, for the streaking a spherical particle (bead) is used. Thestreaking or spreading of a sample onto for instance as a glass slidecan be accomplished by various means, for instance using a rod or abead. Especially when streaking for single colonies, an optimal contactsurface between particle and solid growth medium is desirable.

The bead diameter can be chosen according to specific circumstances. Itmay depend on the desired sample volume to be streaked and/or the samplecharacteristics, such as the (suspected) concentration of microorganism.Suitable beads have a diameter of from about 1 to about 10 mm,preferably 2 to 8. Very good spreading results were obtained with beadshaving a diameter of 4-6 mm. The density of the bead also influences thespreading properties. For example, if the density is too low thefriction with the surface (e.g. an agar culture medium) is not enough toensure good rolling of the bead. On the other hand, a very high densitywill damage the agar surface or even sink into the agar when left at afixed position for a certain time period. Preferably, the density of thebead allows to bead to stay atop of an agar medium for at least 15seconds, more preferably at least 20 seconds. In one embodiment, thebead density is below about 7 g/cm³, preferably below 5 g/cm³. In aspecific aspect, the bead density is less than 4 g/cm³.

The surface of the magnetic particle can be smooth or rough. In oneaspect, the at least one magnetic particle has a smooth surface. In apreferred embodiment, especially for streaking purposes, the at leastone magnetic particle has a rough surface or at least one cavity forreceiving sample. Due to its increased surface area, a rough particlecan accommodate a larger sample volume. It can be a magnetic bead withrandomly or evenly spaced depressions in its surface, e.g. like a golfball.

The mean surface roughness of a material can be expressed by theroughness parameters Ra and/or Rz.

Ra=arithmetical mean roughness value

Ra, signifying the roughness parameter, is a value that is recognizedand used internationally. It denotes the arithmetical mean value of theabsolute values of the profile deviations within the sample area. Thenumerical value of Ra is always smaller than the Rz reading derived fromthe same sample.

Rz=mean roughness depth (peak to valley)

Rz denotes the mean roughness depth, i.e. the arithmetical mean of thehighest single measurements of several single adjacent tracing sections.

In one embodiment, a magnetic particle for use in the present inventionFor instance, has a mean surface roughness in the range of Ra=0.1 μm toRa=25 μm. An Ra value from about 0.1 to about 0.5 μm can be used foronly spreading a sample on a surface. An Ra value from about 10 μm toabout 25 μm is suitably used for inoculation and spreading since a roughsurface picks up a larger inoculum volume. An Ra value of between 2 and15 μm, such as 5-10 μm is preferred. Suitable Rz values range frombetween about 10 to about 40 μm, such as 12-30 μm.

In a specific embodiment, a composite bead has a Ra value of about 6 μmand an Rz value of about 25 μm.

The invention also provides a magnetic composite bead suitable for usein a method of the invention. The composite bead, comprising at leastone magnetic material and at least one non-magnetic material, like ahydrophilic polymer, may have one or more of the above preferredproperties. For example, the invention provides a spherical bead havinga magnetic core, a hydrophilic surface, a diameter of 4-6 mm and/or adensity of less than 6 g/cm³.

Transfer Method

In a magnetically controlled transfer method of the invention, at leastone magnetic particle is used as transport vehicle to transfer at leastpart of the sample, for instance from transferring a liquid sample fromone vial to another, or from a vial to a solid medium. Preferably,multiple particles are used. The particle(s) act(s) as carrier and theamount of sample transferred will depend on the properties of theparticle(s) and of the sample. In particular, the size, shape andsurface of the particle determine the contact surface and the extent ofcohesion with the sample.

The movement of the particle from a first to a second carrier iscontrolled by a moving magnetic field. As a first step, at least part ofsaid sample associated with a first carrier is contacted with at leastone ferromagnetic particle. The term “associated with” is meant toinclude any type of interaction between the sample and the carrier. Forinstance, one or more magnetic particle(s) are added to a liquid sampleheld in a container to absorb or become coated with sample. As anotherexample, a particle is dropped onto a confluent bacterial culture on aculture dish. Optionally, contacting the sample with the particleincludes moving the particle in or on the sample. For instance, one ormore magnetic beads added to a liquid sample are briefly stirred tohomogenize the sample and ensure that a representative portion of thesample becomes associated with the particle. The stirring can beachieved by applying a rapidly changing magnetic field and/or byvortexing. In case a sample is associated with a solid first carrier,the particle may be moved in one or more directions, e.g. by an externalmagnetic field, to increase the contact surface area between theparticle and the sample.

Following contacting the at least one particle with sample, an externalmagnetic field is used to lift the particle coated with sample out of,or away from, the first carrier and transfer to particle to a secondcarrier. Thus, the second step involves applying an external magneticfield to allow for magnetically controlled motion of said particle, suchthat at least part of the sample is transferred to said second carrier.The second carrier can be of any type. It is for instance a liquidculture medium contained in a tube or flask, a solid growth medium, orany solid surface of interest. Again, the magnetic particle may be movedor stirred to maximize sample release from the particle to the secondcarrier. Following sample release to the second carrier, the particlecan be removed or lifted away from the second carrier. It may then bedisposed of or it may be sterilized and re-used.

It will be understood that a transfer method according to the inventionhas endless applications. Specific applications include the preparationof a serial dilution of a stock or concentrated microbial sample. Forexample, a bacterial inoculum held in a first tube is provided with amagnetic bead. The bead coated with inoculum is transferred by a movingmagnetic field into a second tube provided with a liquid broth. Aftervigorous stirring, the bead is again lifted out of the tube andtransferred to a third tube containing a liquid broth. It is of coursealso possible to leave each bead in the tube and use fresh beads foreach transfer. Thereafter, samples may be taken from each tube forfurther transfer and analysis, e.g. for streaking onto a Petri dishplate using a magnetically controlled streaking method according to theinvention. In one embodiment, the invention provides a method fortransfer of a microbial sample from a first carrier to a second carrier,the second carrier being a solid carrier, comprising the steps of:

a) contacting at least part of said sample associated with said firstcarrier with at least one ferromagnetic particle;

b) applying a magnetic field gradient to allow for magneticallycontrolled motion of said particle to said second carrier, such that atleast part of the sample is transferred to the surface said secondcarrier; and

c) applying a magnetic field gradient to allow for magneticallycontrolled motion of said particle on said surface, such that at leastpart of the sample is streaked onto the solid carrier. To allow foroptimal streaking performance (see herein above), it is preferred thatthe at least one ferromagnetic particle is composite bead.

A method of the invention is not limited to sample transfer followed byspreading onto a surface, but is also suitably used for only sampletransfer to a solid surface. For analyses typically performed in(medical) micro-biological laboratories, the inoculum is usually spreadonto the surface of a solid growth medium. In other sectors, such as thewater laboratories it is common to inoculate an empty Petri dish withoutagar. After inoculation with an aqueous sample, liquid agar is dispensedon the Petri dish. The liquid agar mixes with the sample andco-solidifies.

Streaking Method

A method for streaking a microbial sample onto a solid carrier includesany type of sample spreading or sample streaking on any type of solidsurface. For instance, a sample is readily streaked on a glass slide forfurther analysis, such as Gram staining or visual inspection. In apreferred embodiment, a method is provided for streaking a microbialsample, preferably a clinical specimen, for single colonies.

At least one ferromagnetic composite bead (see herein above) iscontacted with a solid surface, the contacting being followed orpreceded by providing the particle with at least part of a microbialsample. Streaking of at least part of the sample onto the solid carrieris performed by the application of a moving external magnetic field toallow for magnetically controlled motion of said bead on said surface.In one embodiment, at least one magnetic bead is provided with sampleprior to being contacted with the solid surface. For example, a magneticbead is dropped into or onto a sample to cover its surface with at leastpart of the sample, followed by transfer or movement of the bead to thesolid surface. In fact, this step can be considered as a “transfermethod” described above.

In another aspect, at least one magnetic particle is contacted with thesolid surface prior to being provided with sample. In an exemplaryembodiment, a sample and a particle are deposited in separate steps on asolid surface after which the particle(s) is moved by a magnetic fieldto spread the sample. The sample may be deposited on or next to theparticle, e.g. manually or automatically. After the streaking iscompleted, the at least one particle can be removed from the solidsurface, again using an external magnetic field.

The solid carrier is preferably a solid growth medium contained in ashallow (disposable) dish, like a Petri dish. For microbiology, agarplates are very frequently used. In a specific aspect, the inventionprovides a method for streaking a microbial sample onto a solid growthmedium held in a split-plate Petri dish.

A solid growth medium supports the growth of one or more micro-organismspresent or suspected to be present in the sample. Various growth mediaare known in the art of microbiology. These include so-called selectivemedia, differential media and enriched media.

Selective media are used for the growth of only select micro-organisms.For example, if a micro-organism is resistant to a certain antibiotic,such as ampicillin or tetracycline, then that antibiotic can be added tothe medium in order to prevent other cells, which do not possess theresistance, from growing. Selective growth media are also used in cellculture to ensure the survival or proliferation of cells with certainproperties, such as antibiotic resistance or the ability to synthesize acertain metabolite. Normally, the presence of a specific gene or anallele of a gene confers upon the cell the ability to grow in theselective medium. In such cases, the gene is termed a marker. Blood agarplates are often used to diagnose infection.

Differential media or indicator media distinguish one micro-organismtype from another growing on the same media. This type of media uses thebiochemical characteristics of a micro-organism growing in the presenceof specific nutrients or indicators (such as neutral red, phenol red,eosin y, or methylene blue) added to the medium to visibly indicate thedefining characteristics of a micro-organism. This type of media is usedfor the detection of micro-organisms and by molecular biologists todetect recombinant strains of bacteria.

Enriched media contain the nutrients required to support the growth of awide variety of organisms, including some of the more fastidious ones.They are commonly used to harvest as many different types of microbes asare present in the specimen. Blood agar is an enriched medium in whichnutritionally rich whole blood supplements the basic nutrients.

Magnetic Field Gradient

A magnetic field gradient is a variation in the magnetic field withrespect to position. Magnetic particles move in the presence of agradient magnetic field. They thus can be made to move along with thefield direction and magnitude. Particles can be navigated or guided e.g.by dragging them using one or more external sweeping permanent magnets.A one-dimensional magnetic field gradient is a variation with respect toone direction, while a two-dimensional gradient is a variation withrespect to two, and so on. The magnetic field can vary in intensityand/or direction and can be achieved by mechanically varying thepositions of one or more magnets with respect to the particle. Suitablemagnets include both permanent magnets and electromagnets. Commerciallyavailable permanent magnets include magnetic metallic elements,composites such as ceramics and ferrites, and rare earth magnets.Electromagnets are also commercially available.

In a preferred embodiment, applying a magnetic field gradient comprisesthe use of an electromagnetic field. The core of the electromagnetpreferably has a high permittivity to achieve a high field strengthusing low electro power.

A specific aspect relates to the use of a grid or array of multiplesmall electromagnets to apply a magnetic field gradient, typically in apredetermined pattern. The strength and direction of the magnetic fieldgradient control the direction and/or magnitude determines the path,lengths, intensity and/or pattern of particle motion. For instance, amagnetic bead coated with sample can be lifted out of a tube by anelectromagnet placed next to the tube to pick up the bead, followed byan upward movement of the magnet that is followed by movement of thebead. The bead can then be transferred to any desired destination whilebeing held by the magnet. Upon arrival at the desired destination, thebead may simply be released from the magnet by switching off themagnetic field.

The skilled person will be able to determine the optimal magnetic fieldstrength for a specific situation. When inoculating and/or streaking asolid growth medium, the field strength has to be as high as possible aslong as the magnetic particle does not damage the medium. In oneembodiment wherein one or more beads are used for sample spreading ontoagar, the magnetic field only produces a horizontal force on the bead(s)and no vertical force, resulting in rolling of the bead(s) withoutdamaging the agar.

Moving the bead can for instance be achieved by using a permanentNeodymium (NdFeB) magnet with a surface field strength of 0.7 T (tesla)and a diameter of 6 mm by 3 mm height. The field strength decreasesrapidly further away from the magnet. The distance between the magneticparticle and the magnet may be minimized in view of the field strengthand/or the accuracy of the magnetically controlled motion. The one ormore magnets are for instance placed essentially directly (e.g. with aspacing of only 0.5 to 5 mm) below the Petri dish. In a streaking methodprovided herein, the external magnetic field gradient can make the atleast one magnetic particle to follow a zig-zag pattern, a concentricpattern, a four quadrant pattern or an at random pattern on the surfaceof a solid carrier (see FIG. 2).

Container

To detect micro-organisms, e.g. bacteria, in clinical samples, aspecimen can be collected from the infected site using a swab which isthen inserted into a transport container. Samples from urine and otherfluids are collected in proper vials.

A further aspect of the invention relates to a container suitable ordesigned for receiving or collecting a microbial sample, said containercomprising at least one magnetic particle. In one embodiment, thecontainer is a disposable container, preferably a disposable test-tubeor vial. Preferably, the container can receive a liquid sample, forinstance a liquid microbial sample, a liquid transport medium and/or aliquid growth medium. It may be provided with a removable cap. Exemplarycontainers include devices for collecting, transporting and/or storingbiological specimens. The at least one magnetic particle present in thecontainer is advantageously used to homogenize and/or transfer themicrobial sample held in the container.

In one embodiment, the container comprises in addition to one or moremagnetic particles, a liquid transport medium or a liquid culturemedium. Such a container is advantageously used as collection device ina culture swab system. Alternatively, or additionally, it may contain aswab for collecting a microbial sample (see e.g. WO2004/086979). Anideal culture swab system must have the ability to absorb organisms fromthe site of infection, to maintain the viability of organisms duringtransport and prior to plating, and finally, to allow the release oforganisms from the swab onto the appropriate media. These are allcritical aspects to be considered when choosing the most appropriatecollection device. The magnetic particles in a container of theinvention can assist the release of sample from the swab prior toanalysis. The container comprising a microbial sample is advantageouslysubjected to a transfer or streaking method as provided herein.

The invention also relates to an apparatus suitable for use in a methodaccording to the invention, said apparatus comprising a culture dishloading station, a sampling unit for applying or inoculating sample ontoa culture dish, a streaking mechanism and a computer control system,characterized in that the sampling unit and/or said streaking mechanismare provided with at least one magnet for generating a magnetic fieldgradient and wherein the control system is connected to said at leastone magnet.

In a specific aspect, the invention provides an apparatus for automatedor semi-automated high-throughput streaking of a microbial sample bymaking use of a magnetic field gradient. The apparatus preferablycontains multiple inoculation and spreading positions to allow forparallel processing of multiple samples. Automated streaking systems perse are known in the art. Typically, they comprise a culture dish loadingstation, a sampling unit for applying or inoculating a sample onto aculture dish, a streaking mechanism and a computer control system forcontrolling the elements of the apparatus. In one embodiment, anapparatus according to the present invention is characterized in thatthe sampling unit, and preferably also the streaking mechanism,comprises at least one magnet for generating a magnetic field gradientand a control system connected to said at least one magnet, in order tomagnetically control inoculation and preferably also the streaking of aculture dish. Accordingly, the invention provides an apparatus suitablefor conducting a method according to claim 1 and those depending thereonin an automated or semi-automated fashion, said apparatus comprising aculture dish loading station, a sampling unit for applying orinoculating sample onto a culture dish, a streaking mechanism and acomputer control system, characterized in that said sampling unit, andoptionally also said streaking mechanism, is/are provided with at leastone magnet capable of generating a magnetic field gradient and whereinthe control system is connected to said at least one magnet.

In another embodiment, the invention provides a streaking apparatus forconducting a streaking method according to claim 2 in an automated orsemi-automated fashion, the apparatus comprising a culture dish loadingstation, a sampling unit for applying or inoculating sample onto aculture dish, a streaking mechanism and a computer control system,wherein said streaking mechanism is provided with at least one magnetcapable of generating a magnetic field gradient and wherein the controlsystem is connected to said at least one magnet, said apparatus furthercomprising a magnetic particle dispensing unit comprising a plurality ofmagnetic composite beads.

Such an apparatus provides a versatile system for varying the streakingprocedure, e.g. in accordance with the sample to be streaked. Thesepatterns can provide an increasing dilution of the sample and areeffected by a magnetically controlled streaking tool. Once so streaked,the prepared dishes can then be incubated to promote growth of one ormore micro-organisms. The growth can then be examined or subjected tofurther tests for isolation and/or identification of the micro-organismtype(s) present in the specimen.

An apparatus provided herein has several advantages over the knownautomated streaking systems known in the art. It is more flexible withrespect to both the specimen type and the type of carrier to whichsample is transferred or spread (e.g. regular Petri dish, miniaturePetri dish, split-type Petri dish, glass slide, culture tube). Differentsizes of particles can be used for different application. Within anapparatus, the magnetic particles are easy to apply to and remove fromone carrier to another due to magnetic control. For reasons explainedherein above, magnetic composite beads are preferred. The particles canbe reused after sterilization. Using magnetic field gradients, anendless number of different streaking patterns can be programmed, andthese can be readily changed in between samples. The use of severalmagnets capable of inducing a magnetic field gradient allows for theprocessing of many samples simultaneously. The overall capacity of amagnetically controlled streaking apparatus can be very high; up to 3-or even 4-fold higher as compared to the best performing apparatuspresently available on the market.

An apparatus of the invention preferably comprises a conveyor system fortransporting and handling containers with specimen (e.g. a tube withurine or a swab) into the apparatus. The containers may be graspedmanually or by an automated manipulating device and move it to a desiredposition, for example next to the culture dish it must be streaked on.The manipulating device may be designed to remove a cap from thecontainer. According to the invention, sample inoculation onto multiplecarriers, e.g. a Petri dish, a tube containing liquid growth broth and aglass slide, can be performed in a single process step, for exampleusing proportional pipetting.

In the sampling unit, sample can be transferred from the container to acarrier, like a culture dish, in an automated fashion. In oneembodiment, the container with specimen is a container according to thepresent invention, comprising at least one magnetic particle, such asone or more magnetic (composite) beads. By adding magnetic particles toa sample in liquid phase, for example urine or to a tube with a swabinserted into liquid transport medium, culture dishes can be inoculatedand spread using the same underlying principle. i.e. throughmagnetically controlled motion of one or more magnetic particles. Thisomits the use of swabs and inoculating loops.

The control system of the apparatus can be programmed to produce amagnetic field gradient that allows for magnetically controlled transferof sample according to a “transfer method” provided herein. Thus, thesampling unit may comprise one or more magnets to allow for 1) retrievalof at least one magnetic particle carrying sample from a specimencontainer, and 2) transfer of said particle and associated sample to adeposit location on a culture dish. The specimen container may then beprovided again with the cap. However, while the container is uncapped,it may be convenient to transfer sample not only to a culture dish, butalso to other types of carriers, such as a glass slide or a liquidbroth. The apparatus may thus also provide for a slide dispensing unitand/or a tube dispensing unit.

Once a sample is transferred to the culture dish, e.g. by means ofmagnetically controlled motion of a particle provided with sample,streaking is effected in the streaking unit of the apparatus. Saidstreaking unit is controlled by the computer control system. If thestreaking mechanism is provided with at least one magnet, it providesfor magnetically controlled streaking bacterial samples in programmablepatterns to produce isolated bacterial colonies.

The apparatus may also provide for other means known in the art to be ofuse in automated sample handling and/or streaking, for example one ormore of the elements described in WO2005/071055 other than the specific“comb-like” streaking device mentioned therein. These include cultureplate lid removal means to remove and replace a lid of the cultureplates to be streaked. Other useful elements of an apparatus include asample identification unit, preferably capable of reading barcodes on aspecimen container.

An apparatus of the invention may be used as follows:

1. a barcoded sample is scanned so the apparatus knows which Petridishes/tube/slice needs to be inoculated. The required Petri dishes arerequested.

2. The Petri dishes arrive at the sampling unit in the correct order.

3. automated lid removal means takes the lid of the dishes

4. a slide dispensing unit and a tube dispensing unit present therequired slides and tubes at the sampling unit.

5. a laboratory worker takes the sample and manually inoculates thePetri dishes, slices and tubes.

6. a magnetic particle dispensing unit places at least one magneticcomposite bead at a predetermined location on the Petri dish

7. the lid is replaced onto the dish and the dish is moved to thestreaking mechanism

8. a magnetic field gradient is applied to spread the sample onto thesolid growth medium in the Petri dish in a desired pattern according tothe sample.

9. After removing of the bead the plates are removed from the streakingmechanism and transported to the incubators to promote growth ofmicro-organisms.

DETAILED DESCRIPTION EXAMPLES Example 1

Experiments were performed to assess the performance of the magneticallycontrolled spreading technique according to the invention as compared tothe conventional spreading technique using a plastic inoculating loop.

Ten (10) μl of a bacterial suspension containing E. coli was applied inthe middle of a Petri dish containing solid blood agar growth medium.One plate was streaked with an inoculating loop in the typical zig-zagstreak pattern. To the second plate, a magnetic bead was applied ontothe inoculated sample. A permanent magnet was placed under the Petridish and the magnet was moved manually such that the bead was moved ontothe solid surface of the growth medium in a zig-zag streak patternsimilar to that of the first plate. Following streaking, the bead wasremoved by the application of a magnetic force. The plates wereincubated for 24 hours at 37° C. to allow for growth of E. colicolonies.

Visual inspection of the plates after the incubation period revealedthat the results of the second plate were superior in the sense that thefinal streak produced more single isolated colonies. Conceivably, thisis due the facts that the beads are rolling while spreading the bacteriaonto the solid medium, providing a better sample dilution than what canbe obtained using conventional loops. Similar advantages of thebead-mediated spreading were observed for the different bacterial strain(S. saprophyticus) and with a different streaking pattern.

Example 2 Magnetic Bead Properties

1. Bead Materials

The principle of spreading with a bead relies on a magneticbead/particle. It is clear that at least one material in the bead shouldbe magnetic or capable of being influenced by a magnet. It wasdiscovered that the bead must roll in order to achieve a good spreadingresult. A bead that is a magnet by itself is therefore not usable. Alsoa ferromagnetic bead which forms a dipole is not usable. The spreadingperformance of various composite beads (numbers 2-5; see FIG. 3) wasdetermined and compared with a non-composite stainless steel bead(number 1) used in the prior art streaking methods and devices.

-   1. Non-composite: a massive stainless steel bead (comparative    example).-   2. Composite (Massive core coated with polymer): a small stainless    steel bead is covered with epoxy to let it roll better.-   3. Composite (Wire core coated with polymer): Steel wire is folded    to a globular shape, the surface is created with epoxy.-   4. Composite (Polymer core coated with magnetic material): An epoxy    bead is covered with a layer of iron particles.-   5. Composite (Homogenous mixture of magnetic and non-magnetic    material): epoxy is mixed with iron particles.    Test Results

In the table below a summary of the test results are visible. The beadsare judged by the following points.

-   Bead number: construction and materials of the bead, see above.-   Rolling: quality of rolling-   Sample pick up: The total inoculated sample volume that can be    picked up by the bead.-   Single colonies: Amount of single colonies on an agar plate, an    indicator for the quality of the bacterial spread on the plate.-   Speed: The speed of the spreading that can be reached.

The points to scored range between − and ++. To determine the totalamount all the “+” are counted. A “−” subtracts a point and “+/−” meansno points.

TABLE 1 test results obtained with non-composite beads versus varioustypes of composite beads. Bead Sample Single number Rolling pick-upcolonies Speed Total 1 − + − + 0 2 +/− ++ + +/− 3 3 + ++ ++ + 6 4+/− + + − 1 5 ++ ++ ++ + 7

The surface of the beads determines among others the quality of thespreading, because the surface is responsible for picking up and losingthe bacteria. The beads with a polymer coating on the outside gives thebest spreading result, whereas the beads with iron on the outside givesa poorer spread. The polymer surface has a greater roughness, so it willpick more sample.

There are two main points that determine the rolling of the bead;

The surface roughness gives resistance with the agar. Through theresistance the bead will encounter a force that will let it roll. Theproperties of the magnetic material. The magnetic properties areresponsible for two forces:

-   1. The forming of a dipole, the dipole will try to stay aligned with    the magnet under the dish-   2. The magnetic field will introduce eddy currents in the metal    leading to a moment opposed to the direction of movement. This force    is only present if the beads roll, it will slow the bead down but    will not prevent rolling.

As long as the force from the resistance is higher than the 2 magneticforces combined, the bead will roll. A preferred magnetic core for thebead is made of a material that has no magnetic dipole and eddycurrents. This is a material with very low demagnetisation forces,divided into small particles (the smaller the particles the smaller theeddy currents). Powdered magnetic material is suitably used for thispurpose.

The massive stainless steel bead known in the art has too high magneticforces and will roll poorly or not at all. The wire bead (no. 4) and thepowder mixed bead (no. 5) give the best results. However, the mixedpowder bead is much easier to produce.

Filler Material.

To determine the suitable amount of iron particles in the bead, thefollowing beads were made

0.1 gram iron, 60% mass, 21% volume 0.2 gram iron, 80% mass, 42% volume0.3 gram iron, 90% mass, 63% volume

The beads with 0.3 gram iron are difficult to produce. The amount offiller material his a slight influence on the spreading quality and aslight influence on the spreading speed. With higher amounts of iron thereachable spreading speed is higher.

The most optimum amount of iron is 0.2 gram. A bead with 0.2 gram ironpowder was therefore used for further testing.

2 Bead Density

Besides the rolling properties, the weight of the bead is alsoimportant. The weight determines the time the bead can lay on the agarwithout sinking. During the automated spreading, it is possible that thebead stays on the same spot for 20 seconds or even longer. The testresults obtained with various types of beads are given in Table 2:

TABLE 2 Effect of bead diameter and bead type on sinking into solidagar. Bead diameter Bead type Time without (mm) (see Table 1) sinking(sec) 5 1 10 5 3 17 5 5 25 7 1 15 7 3 27 7 5 40

The massive steel bead sinks into the agar too fast. The largerpolymer-covered wire bead (no. 3) can reach 20 seconds without sinking.The bead made of polymer mixed with iron powder can stay on the agar formore than 20 seconds, irrespective of the size.

The densities of the materials used are:

Type 1 7.2 g/cm³ Type 3 4.3 g/cm³ Type 5 3.9 g/cm³

These data, combined with the sinking results of table 2 indicate that abead material most preferably has a density below 4.0 g/cm³.

3 Bead size

The bead size has a major influence on the spreading itself; it has aminor influence on the bacterial growth result of the spreading. Withmost bead sizes, e.g. between 4 and 7 mm, good bacterial growth resultsare achieved. The quality of the bacterial growth is determined on theamount of single colonies, the more single colonies there are on aplate, the better.

TABLE 3 Effect of bead diameter on spreading performance. Bead sizeSpeed Sample pick-up Single colonies Sensitivity Total 3 + +/− +/− − 0 4++ + + +/− 4 5 ++ ++ ++ + 7 6 + ++ ++ + 6 7 + + ++ ++ 6 8 +/− + + ++ 4

-   Legend:-   Bead size: the diameter of the beads in mm.-   Speed: the reachable speed of the spreading.-   Sample pick up: The amount the total inoculated sample that can be    picked up by the bead-   Single colonies: Amount of single colonies on an agar plate-   Sensitivity: How difficult is it to stop the bead from spreading    through imperfectness on the agar surface (e.g. bumps, holes, slimy    sample)    4 Bead Surface

The surface of the bead is responsible for the rolling and spreading ofthe sample. The influences on the rolling is discussed in the previouschapter.

Sample Spreading

The surface roughness influences the amount of sample that can be pickedup by the bead. The sample forms a film around the bead. The larger thesurface area, the more sample can be provided onto the bead.

Roughness

Highly suitable beads have the following roughness:

VDI 36: Ra 6.30 μm

-   -   Rz 24.0 μm        (VDI is a roughness indicator used in the mould making industry,        especially in Germany. Ra and RZ values are international        recognized units for surface roughness.)        Hydrophilic/Hydrophobic

The bead surface must be capable of attracting or adsorbing the sample.Almost all biological samples have water as main component. Therefore itis preferred that the bead surface has a hydrophilic character.

5 Production Bead

Materials

For the testing of Type 5 composite beads, a homogenous mixture of homemade iron powder and epoxy was used. These materials are not verysuitable for mass production. Commercially available magnetic fillermaterials can be combined with several polymers include magnetite andferrite. The melted polymer+magnetic particles must flow into a mouldingcavity to make the bead. The particle size of the magnetic material inparticulate form has a significant impact on the flow rate and on thequality of the produced bead. Preferably it should be as small aspossible. Powdered magnetic materials are preferred.

Magnetite: It is a ceramic material and therefore resistant against alforms of corrosion.

Properties

-   -   Material Fe₃O₄    -   Density 5.17 g/cm³    -   Chemical Resistance very high

Ferrite:

Properties

-   -   Material Fe        -   Maximal 0.05% of other materials    -   Density 5.17 g/cm³

The polymer used for binding the magnetic particles/powder may beselected based on the following properties

-   -   High chemical resistance    -   Mixable with Magnetite and Ferrite    -   Mechanical strength up to 120 degrees Celcius (autoclave        temperature)    -   Contains no chemicals ore parts that can come out of the        material into the sample.    -   Low cost price and easy to produce.

The material used for testing is

-   -   Material PA, Polyamide (nylon)    -   Density 1.39-1.58 g/cm³    -   Tensile strength 70-120 MPa    -   Useful temp range −50-120° C.    -   Melting Temp. (range) 216    -   Hardness 92 Rockwell (E Scale)    -   Chemical Reactivity low    -   Chemical Resistance high

Other possibilities are

-   -   Common thermoplastic materials which meet the requirements as        stated above. Example:    -   Material PP, Polypropylene    -   Density 0.90 g/cm³    -   Tensile @ Yield 23 MPa    -   Softening Temp. 143° C.    -   Melting Temp. (range) 160° C.-166° C.    -   Hardness 66 Rockwell (R Scale)    -   Chemical Reactivity low    -   Chemical Resistance high

The amount of polymer in comparison to the amount magnetic material is:

20-25% polymer (binding)

75-80% magnetic material (magnetic strength)

The invention claimed is:
 1. A method for transfer of a microbial samplefrom a first carrier to a second carrier, comprising: contacting atleast part of said sample associated with said first carrier with atleast one ferromagnetic particle, the particle comprising a compositebead having a surface roughness (Ra) in the range of 0.1 to 25 μm, adiameter of between 2 to 8 mm and a density below 7 g/cm³; and applyinga magnetic field gradient to allow for magnetically controlled motion ofsaid particle to said second carrier, such that at least part of thesample is transferred to said second carrier.
 2. The method of claim 1,wherein said composite bead comprises a hydrophilic surface.
 3. Themethod of claim 1, wherein said at least one composite bead comprises atleast one polymer and at least one magnetic material.
 4. The method ofclaim 1, wherein the composite bead is comprised of a material selectedfrom the group consisting of magnetite, ferrite, and combinations of anythereof.
 5. The method of claim 1, wherein the composite bead comprisesa polymer that is heat resistant up to about 120° C.